There is widespread use of extracts of plants, and tinctures (extracts of plants dissolved in an organic solvent, usually ethanol) of plants as natural health remedies. Extracts of plants are particularly desirable, for example, as the quantity to be ingested is decreased relative to using fresh plant material, and also the storage life is improved relative to the plant material. There is a desire to standardize the strength of the plant extracts against “marker compounds” with known or suspected bioactivity. The solvents most widely used to extract these plant materials are water and water+ethanol mixtures and to a lesser extent, water with other hydrophilic organic solvents, such as acetone and acetic acid. These solvent mixtures have high solvent strength but low selectivity, resulting in the extraction of all the desired physiologically active components and also a large amount of inactive or even undesirable components. There exists a need, therefore, for a separation process that can further fractionate the crude extracts obtained from plants using aqueous-organic solvent mixtures, to be able to increase the concentration of the desired bioactive or marker compounds and reduce or remove inactive and/or undesirable components.
The most common solvents for extracting plant materials to obtain biologically active extracts are mixtures of water and ethanol. The proportions can vary from 0 to 100% of each solvent component. These solvent mixtures are widely used, as they require relatively simple technology to perform extractions from plant material, and are acceptable for food grade processes. Other alcohols, such as isopropanol and propanol can be substituted for ethanol but the allowable concentrations in the final product are diminished relative to ethanol. The aqueous-organic solvent mixtures are not limited to water and ethanol mixtures. Numerous other hydrophilic organic solvents, such as acetone, acetic acid, dimethylsulfoxide and methanol may also be used.
Near-critical or supercritical CO2 is an alternative food-grade solvent possibility for the extraction of biologically active compounds because its critical temperature and pressure (31° C., 74 bar) is attained relatively easily and, furthermore, CO2 is inert, non-toxic, cheap and readily available. It is well known in the art that CO2 is a substantially weaker solvent than ethanol. The range of extractable physiologically active compounds using supercritical CO2 is limited in general to lipophilic (fat soluble), low to medium molecular mass molecules, such as flavour and fragrance compounds as described in U.S. Pat. No. 4,632,837. Attempts to improve the solvent power have been described in, for example, U.S. Pat. No. 5,017,397 where pressures greater than 350 bar have been used, and U.S. Pat. No. 5,252,729 where plant material and CO2 insoluble molecules have been hydrolysed to break the molecules into extractable fragments.
The extraction of natural products from plant materials using a single phase mixture of CO2 and an organic solvent, known as a co-solvent, has been attempted to try and overcome the limitations of pure CO2. The co-solvent is usually used in a proportion of 5 to 20% by mass. Examples of patents that describe the use of CO2 and a co-solvent for the extraction of physiologically active compounds from plants include U.S. Pat. No. 5,252,729 (referred to above), U.S. Pat. No. 6,569,640, and U.S. Pat. No. 6,291,241. In addition, U.S. Pat. No. 6,326,504 describes a method for extracting plant material using an organic solvent with a near-critical fluid dissolved in it, where the near-critical fluid is less than 50%, and preferably between 7 to 26%, by weight of the total solvent mixture. Extraction is carried out at a pressure of 50-500 bar, and preferably 150-280 bar. The purported advantages are a reduction in viscosity and increase in diffusivity of the solvent mixture. A reduction in the solvent power of the mixture relative to the pure organic solvent is not noted, but is known in the art as described below. All of the aforementioned processes require the use of high pressure in the initial extraction of the plant material, which in turn requires large extraction vessel volumes. This gives rise to high capital costs of processing plant.
An alternative near-critical fluid to CO2 is dimethyl ether. Dimethyl ether with and without water has also been used to extract plant and animal materials, as described in U.S. Pat. No. 4,234,619, U.S. Pat. No. 4,157,404, U.S. Pat. No. 4,136,065, U.S. Pat. No. 4,069,351, U.S. Pat. No. 4,048,343, U.S. Pat. No. 3,900,288, U.S. Pat. No. 3,795,750, and JP 2001106636. However, none of the aforementioned patents describes a method for the extraction and fractionation of extracts of plants or animal products contained within an aqueous-organic solvent solution. Much lower pressures can be used to extract a wider variety of compounds with dimethyl ether compared to supercritical CO2, and drying of the plant material may not be required. However, large pressurized extractors are still required, along with large volumes of dimethyl ether, which can be a safety hazard. The range of compounds that can be extracted is also substantially less than aqueous-organic solvents.
The poor solvent properties of CO2 with respect to polar and/or large molecules can be used to produce fine powders. The CO2 GAS (Gas Anti-Solvent) precipitation process was first described in U.S. Pat. No. 5,360,478. A solid material can be recrystallised from systems comprising:                (i) a solute, which is the eventual material recrystallised;        (ii) a liquid, which is a suitable solvent for the solute; and        (iii) a gaseous component (CO2), which is soluble in the solvent and causes the solvent to approach or attain a supersaturated state, thereby precipitating the solute material.        
A variation on the GAS process is the SAS (Supercritical Anti-Solvent) or PCA (Precipitation with a Compressed Anti-Solvent). Here, the organic solvent solution containing the solute to be recrystallised is mixed with a supercritical fluid under conditions where the solvent is completely miscible with the fluid, but the solute is insoluble in the new solvent mixture (WO 9003782). The solute precipitates to form a fine powder.
U.S. Pat. No. 5,349,084 describes a process for the purification of dicarboxylic acids using GAS fractionation following the production of crude acids in a fermentation process, extraction of the acids from the fermentation broth with hot water, reduction of the water content in the crude acid/water mixture to 3-30%, followed by re-extraction of the acids with an organic solvent. CO2 is used as an anti-solvent to precipitate unwanted compounds from solution. The pressure of the anti-solvent is below the critical point for CO2.
JP 6048952 describes a multi-step process where plant material is extracted with an organic solvent (usually ethanol) to yield a solution containing a fat soluble physiologically active component. The solution is mixed with a high pressure gas (CO2) under conditions where the gas dissolves to a large extent in the liquid solvent to precipitate polar compounds and/or a polar liquid fraction. The organic solution containing dissolved CO2 is then compressed to a pressure of 100-500 bar and mixed with further (supercritical) CO2 at the same pressure to dissolve further CO2 into the organic solvent to further reduce the solvent power of the organic solvent. The resultant solution is then passed into a column. The medium polarity component is precipitated into the base of the column, and the low polarity component remains dissolved in the new solution of organic solvent and CO2. This solution then passes through a pressure reduction valve and into a separator where the CO2 is separated from the organic solvent and low polarity component. The CO2 then passes through a further separator, under conditions where CO2 is a gas, to remove contaminants.
However, the process of JP 6048952 suffers from a number of disadvantages. The first disadvantage is that the first separation step results in a gas saturated organic solution that must be pumped to a higher pressure. This results in cavitation in the pump due to the release of gas. The second disadvantage is that CO2 gas has very poor solubility in aqueous-organic solutions, even at high pressures (the document states that an aqueous-ethanol solution may be used in the process, although no example is provided). The process is therefore limited to low water levels when the solvent is aqueous-organic. The third disadvantage is that recycling CO2 requires a gas compressor, due to the low pressure requirements for the first separation stage. The use of a gas compressor requires more energy than a pump.
A supercritical anti-solvent process for the fractionation of propolis tinctures is disclosed in O J Catchpole, J B Grey, K A Mitchell, J S Lan, J. Supercritical Fluids, 29, 97-106, 2004. Bees collect a resinous exudate from the leaves of some species of trees, and then mix this with beeswax to obtain propolis, which is then used to provide protection for the hive. Propolis is a complex resinous mixture of components including waxes, flavonoids, and detritus from the hive. The propolis is scraped from the hive, and a tincture is made by dissolving the propolis in ethanol or an ethanol and water mixture. This tincture is then further processed to remove waxes and detritus. The process described identifies conditions under which aglycone flavonoids (flavonoids without attached carbohydrate groups) can be extracted from the tincture using supercritical CO2 and other unidentified components can be precipitated. The process is efficient at recovering aglycone flavonoids from tinctures made using 95% ethanol, but the efficiency is decreased markedly when the ethanol content of the ethanol-water mixture is decreased to 70%, and water increased to 30%. There is no description of a process for the fractionation of plant or animal material that has been directly extracted with an aqueous-organic solution, nor a process for isolating chemical compounds other than aglycone flavonoids. Aglycone flavonoids are not normally found in hydroalcoholic extracts of leaf material, but much more polar glycosylated flavonoids can be found.
WO 2005/075614 describes a process for the extraction of olive leaf, followed by fractionation of the extract. Here the olive leaf is extracted either with hexane or ethanol, and then the crude extract has the majority of the solvent removed by vacuum fractionation and is filtered or clarified to remove precipitates. Hexane extracts only low polar compounds, while ethanol extracts low to medium polarity compounds from the leaf. The concentrated extract is then contacted with supercritical CO2 in a countercurrent packed column. A second, more polar organic solvent, up to a concentration of 10%, is added to the supercritical CO2 to improve the solubility of low to medium polarity compounds. The CO2 and organic co-solvent extracts all the remaining solvent and the low to medium polarity compounds in the extract, which can be recovered by two stage pressure reduction. Highly polar compounds are precipitated inside the column when using ethanol. The method for recovering these compounds is not described. A precipitate fraction is not obtained when using hexane as the primary extraction solvent. There is no description of a process in which the plant material is extracted with an aqueous-organic solvent mixture containing more than 5% water, nor a process in which the aqueous-organic mixture is contacted directly with the near-critical fluid (the solvent mixture is first partially evaporated).
The process described in WO 2005/075614 has several disadvantages. Firstly, the crude extract must be partially evaporated and filtered/clarified. This requires high energy costs to remove and recover the solvent, and requires more processing steps and process equipment. The most polar compounds are not extracted in the solvent extraction step because an aqueous-organic solvent has not been used. The highest polarity compounds that have been extracted are precipitated inside the column and can only be removed in a discontinuous manner by removing CO2 from the column, and then cleaning the column with an organic solvent. A further disadvantage is that a second organic solvent is required as a co-solvent to increase the solvent power of CO2 for low to medium polarity compounds.
The inventor has now made the surprising finding that a process for fractionating the constituents of a solution (obtained by extracting plant or animal material with an aqueous-organic solvent) by contacting the solution with a near-critical fluid followed by further processing overcomes or ameliorates one or more disadvantages of known processes.
It is therefore an object of the invention to provide a process for fractionating the constituents of a solution of components extracted from plant or animal material, or at least to provide a useful alternative process.